Bioluminescent bioassay system

ABSTRACT

A system for measuring toxicity levels of a solution includes a water proof sample container transparent to visible light which holds an aqueous test solution containing bioluminescent organisms. A light tight chamber has a cavity which holds the sample container and includes a light port. A stress generating system positioned in the sample container generates pressure pulses which stimulate the organisms to generate light emissions. A light detector system mounted to the light tight chamber in a light tight manner detects light emissions generated in the sample container which propagate through the light port and are received by the light detector system. The light detector system generates an electric pulse in response to detecting each detected light emission. A controller enables the stress generating system and the light detector system, and then counts the electric pulses within a predetermined period of time.

STATEMENT OF GOVERNMENT INTEREST

The invention described herein may be manufactured and used by or forthe Government of the United States of America for governmental purposeswithout the payment of any royalties thereon or therefor.

BACKGROUND OF THE INVENTION

The present invention relates to the field of detecting bioluminescentemissions and, more particularly, to counting photonic emissions of anaqueous solution of bioluminescent organisms to determine toxicitylevels of the solution.

Bioluminescence is a visible blue light produced either intermittentlyor continuously by numerous terrestrial and aquatic organisms. Manymarine dinoflagellate species are able to produce bioluminescence aspart of their daily physiological processes. Similarly, some marinebacteria are also bioluminescent. Since various toxicants are known toreduce the light intensity output of bioluminescent bacterial cultures,they have been used as test organisms to detect the toxicity ofatmospheric samples, herbicides, and some chemicals.

Phytoplankton bioassays are effective biological tools to assessenvironmental contamination because they are primary producers in thefood chain, and their inherent sensitivity to toxic chemicals.Phytoplankton bioassays tend to be simple, rapid, and inexpensive whencompared to more complicated and involved assays that use fish orinvertebrate species. Phytoplankton bioassays generally involve theenumeration of phytoplankton cells to determine stress in algalpopulations when exposed to a single toxicant or chemical mixtures.These assays have been successful, but tend to be labor intensive.

U.S. Pat. No. 4,950,594, "Microbiological Assay Using BioluminescentOrganism" describes a method for assaying drilling fluids for toxicity.The method consists of agitating an aqueous solution of bioluminescentorganisms, Pyrocystis lunula, using a stirring rod fitted into the chuckof a variable speed motor. The rod is stirred at approximately 100 rpmto stimulate the organisms to luminesce. Bioluminescence is measuredwith a solid state photometer circuit as described in U.S. Pat. No.4,689,305 which provides an output current proportional to the lightdetected by an integrated photodetection assembly. Since the '305circuit operates in a current mode, the integrated photodetectionassembly only detects average intensity. The circuit is not sensitiveenough to detect individual photons generated by the organisms.

U.S. Pat. No. 4,563,331, "System For Measuring Bioluminescence FlashKinetics" describes a system for detecting and measuring bioluminescentsignatures of planktonic organisms. The system includes a light tightchamber in which is positioned an organism sample holder containingfiltered seawater and bioluminescent organisms. Photomultiplier tubesare mounted to the light tight chamber to detect any light generated bythe organisms. The organisms are stimulated to luminesce by a vacuumpump which draws the seawater through a filter from the bottom of thesample holder. Signals generated by the photomultiplier tubes inresponse to detecting the light emissions from the organisms areprovided to Davidson multichannel analyzers.

In the operation of the '331 system, the vacuum pump draws seawaterthrough the filter at the bottom of the sample holder. The suctioncauses the organism to be drawn against the inlet side of the filter,where they concentrate. This tends to damage the organisms. Moreover,light generated by organisms squeezed between other organisms is notdetected by the photomultiplier tubes, and further, the amount of lightthey produce can be highly variable. Variations in time delays betweenenergizing the vacuum pump and initiating data collection betweenexperiments as a result of manual operation of the '331 systemintroduces a variable in the experimental results. This variable makesit difficult to relate the results of one experiment to another,negatively impacting the repeatability of experiments performed usingthe '331 system.

Therefore, there is a need for a bioluminescent assay system in whichthe bioluminescent organisms may be stimulated without being damaged,and in which the stimulus is consistent. A further need exists for abioluminescent system which provides good repeatability betweenexperiments.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a bioluminescent bioassay system embodyingvarious features of the present invention.

FIG. 2 is a perspective view of the sample support.

FIG. 3 is an end view of the sample support taken along section 3--3 ofFIG. 2.

FIG. 4 is a perspective view of the light tight chamber.

FIG. 5 is a three-quarter view of the cap and motor assembly.

FIG. 6 is a partial cross-sectional view of the cap and motor assembly.

FIG. 7 is a cross-sectional view of the optical detector system.

FIG. 8 is a flow chart of the operation of the invention.

FIG. 9 is a schematic diagram of a light detector circuit.

FIG. 10 is a schematic diagram the high voltage power control circuit.

SUMMARY OF THE INVENTION

A system for measuring toxicity levels of a solution includes a waterproof sample container transparent to visible light which holds anaqueous test solution containing bioluminescent organisms. A light tightchamber has a cavity which holds the sample container and includes alight port. A stress generating system positioned in the samplecontainer generates pressure pulses which stimulate the organisms togenerate light emissions. A light detector system mounted to the lighttight chamber in a light tight manner detects light emissions generatedin the sample container which propagate through the light port and arereceived by the light detector system. The light detector systemgenerates an electric pulse in response to detecting each detected lightemission. A controller enables the stress generating system and thelight detector system, and then counts the electric pulses within apredetermined period of time.

An important advantage of the present invention is that it provides anautomatic system for determining relative toxicity levels in an aqueoussolution.

A further advantage is that the present invention provides good testresult repeatability with low statistical variance.

Another advantage of the present invention is that it includes ahardware based safety interlock system which prevents the opticaldetector system from being damaged from exposure to ambient light.

These and other advantages will become more apparent upon review of thespecification and drawings herein.

DESCRIPTION OF THE PREFERRED EMBODIMENT

Referring now to FIG. 1, there is shown a partial cross-sectional viewof a bioluminescent assay system 10 for measuring toxicity levels in anaqueous solution containing light emitting organisms. System 10 is shownto include an optically transparent sample container 12 for holding atest sample of an aqueous solution 14 of bioluminescent organisms 16which is to be tested for toxicity. The bioluminescent organisms 16 areselected so as to generate photonic emissions 17, or light whensubjected to pressure pulses, such as turbulence, in the aqueoussolution 14. The sample container 12 is mounted within a cavity 18formed in a light tight chamber 20. Stress generating means forstimulating the organisms 16 to generate photonic emissions (light) 17may include a motor 22 having a motor output shaft 24 on which apropeller 26, or other stirring type of mechanism, such as a paddle, ismounted. The motor 22 is mounted to the top of a cap 25 which fits overthe light tight chamber. The shaft 24 extends into the sample container12 so that the propeller is immersed in the aqueous solution 14.Energizing the motor 22 causes the propeller 26 to stir the aqueoussolution 14, thereby generating shear stresses, or pressure pulses inthe fluid 14 which tend to simulate the organisms 16 to emit light. Themotor 22 is mounted on top 23 of a cap 25 fitted to the upper end of thechamber 20 so as to create a light tight seal so that ambient light doesnot enter the chamber 20. The chamber 20 includes a light port 28through which photonic emissions 17 generated by the organisms propagatethrough light port 28 to an optical detector system 30 mounted in alight tight manner on the chamber 20 which detects any photonicemissions 17 generated by the organisms 16. The optical detector system30 is mounted to the chamber 20 in a manner which prevents ambient lightfrom entering the chamber 20 or reaching the light sensing element 164(FIG. 7) of the optical detector system 30. A controller 32 enables theoptical detector system 30 and the stress generating system forstimulating the organisms 16. In the preferred embodiment, thecontroller 32 may be implemented as an imbedded micro-controller. Asensing circuit, not shown, partially located in the cap 25 provides anoutput signal OPEN to the controller 32 which represents whether the cap25 is properly positioned on top of the chamber 20 so as to preventambient light from reaching the optical detecting system 30. Should thecap 25 be positioned so that ambient light enters the chamber 20, thesignal OPEN is a logic low, causing the controller 32 to disable powerto the photodetecting circuit 30.

The system 10 also includes a start switch 33 coupled to the controller32 which allows the optical detector system 30 and motor 22 to beautomatically enabled in an appropriate time sequence. A second switch37 operably coupled to the controller 32 and to the optical detectorsystem 30 in accordance with well known techniques provides a humanoperator with the option of operating the system 10 in an automatic ormanual mode, or of disabling the system altogether when the switch 37 isplaced in the OFF position. Positioning the switch 37 in the automaticmode enables the motor 22 through the motor control (MTR CNTRL) line,and enables the optical detector system 30 by way of the"high-voltage-low-voltage" (HVLV) signal line. The optical detectorsystem 30 generates an electronic signal pulse in response to receivingeach single photonic emission 17. Each signal pulse is provided to thecontroller 32 via the DATA signal line. When operated in an automatedmode, the controller 32 counts the number of signal pulses generated bythe optical detector system 30 within a predetermined period of time.

The sample container 12, may for example, be a cuvette made of amaterial such as polystyrene which is optically transparent to thewavelengths of photonic emissions of interest generated by thebioluminescent organisms 16. Such wavelengths generally fall within therange of about 450-500 nanometers.

In the presently preferred embodiment, as shown in FIG. 2, a samplesupport 21 is used to support the sample container 12 within the lighttight chamber 20. The sample support 21 may be generally cylindricallyshaped, as shown in FIGS. 2 and 3. Sample support 21 may include acavity 40 extending from a first planar surface 42 through to theopposite planar surface 44 (FIG. 3) generally along the direction of thelongitudinal axis a--a of the sample support 21. The support 21 furtherincludes a light port 46 extending radially outwardly from the cavity 40through to the surface 47 of the sample support 21. By way of example,the cavity 40 preferably may have a cross-sectional area shaped, by wayof example, as a square, which allows the sample container 12 to slideand be supported within the cavity 40 without rotation. However, it isto be understood that the cavity 40 may have a cross-sectional areaconfigured into other shapes, such as circles, triangles, or rectangles.Mounting holes 48 which extend the longitudinal length between theopposed planar surfaces 42 and 44 generally parallel to the axis a--areceive threaded fasteners, not shown, for fastening and indexing thesample support 21 to the chamber 20.

As more clearly seen in FIG. 3, a generally round slot 50 having akeyway 52 extends generally from the surface 42 to the surface 44 downalong the longitudinal length of the sample support 21 and parallel tothe axis a--a so as to be in fluid communication with the cavity 40.Referring also to FIG. 2, a rod 54 having a handle 56 is fitted to slidewithin the generally circular slot 50. A strut 58 extending generallyperpendicular from the rod 54 and through the keyway 52 towards thecavity 40 towards its bottom allows the rod to slide and be retainedwithin the slot 50 by the surface 42 because the keyway 52 extends onlyfrom the bottom surface 44 to within a fixed, discrete distance, as forexample 0.5 inches, from below the surface 42. Thus, the strut 58retains the rod within the slot 50. The combination of the rod 54 andstrut 58 provides a lift mechanism for facilitating removal of thesample container 12 from the cavity 40 by pulling on the handle 56,causing the strut 58 to lift the bottom of the sample container 12 upout of the cavity 40.

The light tight chamber 20, described with reference to FIG. 4, includesa cavity 60 extending along the longitudinal axis b--b between opposedplanar surfaces 61 and 53. At the top of the chamber 20, the cavity 60is defined by an annulus 55 extending up from the planar surface 62. Theannulus 55 includes a groove 57 in which an O-ring 65 is placed. Thecavity 60 is shaped to slidably receive the sample support 21 so thatthe aperture 46 of the sample support 21 may be aligned with a circularaperture 64 generally centered about an axis c--c which preferably isperpendicular to and intersects the longitudinal axis b--b. The alignedapertures 46 and 64 of the sample support 21 and light tight chamber 20,respectively, collectively comprise the light port 28. The aperture 64preferably is sized to receive an optically neutral density filter 118(FIG. 7). The use of the filter 118 extends the dynamic counting rangeof the system. An annular planar surface 66 formed in the chamber 20 andcentered about the aperture 64 provides a mounting surface so that theoptical detector system 30 may be attached to the chamber 20 in a lighttight manner. Three threaded studs 68 extending perpendicularly from thesurface 66 are used to fasten the optical detector system 30 to thechamber 20 as shown in FIG. 1.

By way of example, with reference to FIGS. 4, 5 and 6, collectively, themotor 22 is rigidly mounted to a cap 25 having an annulus 71 with aninternal diametral surface 72 sized to fit over the O-ring 65 supportedin the groove 57 of the annulus 55 on top of the container 20. A slightinterference fit between the internal diametral surface 72 and theO-ring 65 assures a light tight fit between the cap 25 and the chamber20. The motor shaft 24 extends through an aperture, not shown, in thecap 25, and is supported by an annulus 74 extending from the innerplanar surface 76 of the cap. In order to assure that no ambient lightpenetrates between the motor and the cap 25, by way of example, aresilient sealing member 78, such as a gasket or O-ring, may be fittedbetween the motor 22 and the surface 80 of the cap 25. The sealingmember 78 is preferably fitted between an annular groove 84 formed inthe housing of the motor 22 and an annular groove 86 formed in thesurface 80 of the cap 25. The end cap 25, light tight body 10, andsample support 21 are preferably made of a material which is chemicallynonreactive with sea water, as for example, DELRIN®, polycarbonate, orpolystyrene.

An example of one preferred optical detection system 30 is describedwith reference to FIG. 7, where there is shown a cylinder 100 havinginternal threads 102 at ends 104 and 106. An end ring 107 having aflange 105 and an inner annulus 109 with external threads 108 isthreaded into the internal threads 102. An annular land 110 on the innerannulus 109 provides a surface for receiving an O-ring 112. When the endring 107 is threaded into the cylinder 100, the O-ring 112 is compressedbetween the flange 105 and the cylinder end 104 to provide a light tightseal. The end ring 107 also includes an inner aperture 114 and acoterminous outer aperture 117 which define an inner flange 116 at theirinterface. The outer aperture 117 is sized to slidably receive anoptional filter 118 with minimal clearance. The flange 116 provides abackstop for the filter 118. By way of example, the filter 118 may beimplemented as any of an Oriel Model Nos. 59980, 59990, or 59000. Theouter planar surface 120 of the flange 105 has an annular groove 119 forreceiving an O-ring 122 which is compressed to provide a light tightseal when the optical detection assembly is attached to the light tightbody 20. Apertures 113 extending through the flange 105 around its outerperiphery slide over the studs 68 (FIG. 4) to facilitate mounting andattaching the optical detection system 30 to the light tight chamber 20with nuts 43 as shown in FIG. 1.

Still referring to FIG. 7, the optical detection system 30 furtherincludes an end cap 124 having an outer flange 126 and external threads128 formed in an annulus 130. An annular land 132 between the externalthreads 128 and the inner planar surface 134 of the flange 126 supportsan O-ring 132 which is compressed to provide a light tight seal betweenthe end cap 124 and the cylinder 100. A pair of struts 140 extendperpendicularly from the inner surface 146 of the end cap 124. Thestruts 140 may be attached to the end cap 124 using threaded fasteners144 which extend through the end cap and into the struts 140. The ends146 of the struts 140 support a disc shaped mounting plate 148 which isattached to the struts by threaded fasteners 150. The threaded fasteners150 also attach an electrically insulating, concentric ring spacer 152to the end plate 148. The outside diameter of the spacer 152 preferablyis slightly less than the inside diameter of the cylinder 100 so thatthe spacer may minimize any wobbling of the struts 140 and the mountingplate 148 within the cylinder 100. A pair of struts 154 (only one isshown) extend perpendicularly from the surface 156 of the support plate148 from which they are attached with threaded fasteners 158 (only oneis shown). The struts 154 support a socket 160 in which is mounted aphotomultiplier tube 162 having a light sensing element 164, or lens,which receives the photonic emissions 17 generated by the organisms 16.Also supported by the struts 140 is a high voltage (HV) power supply 180which provides power to the photomultiplier tube 162. The HV powersupply 180 may be implemented, for example, as a Venus Scientific, Inc.,Model C30. Electrical coupling link 167 provides a power and data linkbetween the photomultiplier tube 162 and the HV power supply 180.Electrical coupling link 167 provides a power and data link between theHV power supply 180 and electrical socket 168 mounted on the externalplanar surface 170 of the end cap 124. The photomultiplier tube 162 maybe implemented as an RCA, Model 8575 photomultiplier tube ("PMT")operated in the single photon counting mode. The cylinder 100, end ring107, and end cap 124 are preferably made of a ferrous material toprovide a magnetic shield which prevents external magnetic fields frominfluencing the propagation of photoelectrons in the photomultipliertube 162. Such ferrous material should be chemically nonreactive withsea water, and may be for example, stainless steel or μ-metal.

The operation of the system 10 is described with reference to FIG. 8.The system 10 is initialized by providing an input representing thedesired counting time, "TIME" to the controller, 32 at step 200. Thecounting time refers to the period in which electronic pulsescorresponding to the photonic emissions 17 generated by thebioluminescent organisms 16 are to be counted. Next, at step 202, theaqueous sample solution 14 with the bioluminescent organisms 16 may beplaced in the sample holder 12. The sample holder 12 is placed in thesample support 21 which is mounted within the light tight chamber 20. Ahuman operator then mounts the cap 25 on the light tight body 20. Atstep 204, the operation of the controller 32 is initiated by pushing the"START" switch 33 which enables the HV power supply 180 andphotomultiplier tube 162 and initiates a first internal clock, CLOCK 1,in the controller 32. A predetermined period, for example, of fiveseconds elapses in order to stabilize the HV power supply 180. Thecontroller 32 proceeds to enable the HV power supply 180 andphotomultiplier tube 162, as well as the serial port at step 206.

The controller 32 then reads the first clock at step 208. A decision ismade by the controller at step 210 to determine if the time elapsed fromthe initiation of the first clock equals a predetermined value, K If thedetermination at step 210 is "NO", then the controller 32 returns tostep 208. If the determination at step 210 is YES, the controller 32initiates a second clock, CLOCK 2, at step 211 which counts down from atime, d+TIME, where "d" represents a delay, by way of example, of twoseconds to assure that the organisms 16 have not been pre-stimulated.Pre-simulation would tend to invalidate the total pulse count. Ingeneral, the time delay provides a "dark count" which is actuallyinsignificant compared to the photon count generated by stimulatedorganisms (approximately 400 counts out of typically 300,000 to1,000,000 counts). The dark count refers to the number of photonsgenerated by thermionic emissions from the photocathode and dynode chainin the photomultiplier tube 162 and to light generated by the organisms16 when they are not being stimulated. The controller then reads CLOCK 2at step 212. Proceeding to step 214, the controller 32 determines if thetime indicated by the second clock equals zero. If the determination atstep 214 is "NO", the controller 32 enables the stirring motor 22 atstep 216, and then at step 218, allows the controller to count pulsesgenerated by the optical detecting system 30. The controller 32 thenreturns to step 212. If the determination by the controller at step 214is "YES", then at step 220, the controller 32 disables the HV powersupply 180, stirring motor 22, and serial port 35, and ceases to countpulses generated by the optical detecting system 30. The operation ofthe system then returns to step 202.

While CLOCK 2 decrements, any photons emitted by the organisms 16detected by the optical detector system 30 cause the photomultiplier 162to generate an electric pulse which is counted by the controller.Generally, while CLOCK 2 is decrements, output data including elapsedtime and count data are sent to output port 35, such as an RS-232 serialport, of the controller 32. Thus, the data may be sent to a remotecomputer to log and analyze data, or to a peripheral device such as aprinter.

Referring to FIG. 9, the system 10 also includes a light detectorcircuit 300, included within the controller 32, which causes the opticaldetector system 30, and particularly, the photomultiplier 162, to bedisabled in the event that the cap 25 is removed from the light tightbody 20 in order to prevent damaging the photomultiplier 162 fromoptical overload. Once the light detector circuit 300 causes power tothe HV power supply 180 to be turned off, the photomultiplier 162 isdisabled. Power to the HV power supply 180 is not restored until switch37 (FIG. 1) is toggled switched to its "OFF" position and then back toeither its manual or automatic select position, thereby providing thesystem 10 with a safety interlock.

Referring now to FIG. 9, there is shown an example of light detectorcircuit 300 which includes an infrared energy emitting diode CR1 whichis mounted in an arcuate shaped block 61 (FIG. 4) so that it irradiatesinfrared energy through the aperture 59 along a path 63 which isoccluded by cap 25 when the cap is mounted over annulus 67, but whichirradiates the phototransistor Q1 which receives the infrared energythrough aperture 69 when the cap 25 is dislodged or removed from theannulus on top of the light tight body 20. By way of example, thedistance between CR1 and Q1 may be about three inches.

When the cap 25 (FIGS. 5 and 6) is positioned on the annulus 55 (FIG. 4)so that ambient light does not enter the light tight body 20, the gatevoltage of phototransistor Q1 is sufficiently low so that there is verylow conduction from the +5 V supply through resistor R2 down to ground.When the gate voltage of Q1 is low, the input signal, OPEN, at pin 2 ofcomparator LM311N is high, whereupon the output, LID, of the comparatoris a logic high. In other words, when the infrared light path betweendiode CR1 and phototransistor Q1 is blocked, or occluded, i.e., noambient light enters the light tight chamber 20, the output of thecomparator LM311N is a logic high. However, when infrared energygenerated by the diode CR1 is detected by the phototransistor Q1, thegate voltage is sufficient so that the 5V source conducts through thephototransistor Q1 to ground. In such case, the input 2 of LM311N isrelatively low, whereupon the output, LID, of the comparator LM311N is alogic low. Thus, it may be appreciated that the light detecting circuit300 provides an output LID which represents whether ambient light is oris not entering the light tight chamber 20. Output LID is provided to ahigh voltage interlock circuit 182 (FIG. 10) which allows thephotomultiplier tube 162 to be energized only at appropriate times, andto prevent damage to the photomultiplier tube 164. Damage could occur,for example, when the cap 25 is removed from the light tight body 20,whereupon the photomultiplier tube 164 would be saturated andoverwhelmed by excessive light. The high voltage interlock circuit 182is part of the controller 32.

FIG. 10 illustrates an example of a high voltage (HV) interlock circuit("HV circuit") 182 which performs a safety interlock function thatprevents the photomultiplier tube 162 from being energized until theswitch 37 (FIG. 1) has been toggled through an OFF position. By way ofexample, the HV interlock circuit 182 is shown to include fourinterconnected NOR gates U21A, U21B, U21C, and U21D; a flipflop U22A;and an inverting buffer U20. Flipflop U21B receives a normally logichigh HVMAN input at pin 5 and a grounded input at pin 6. Flipflop U21Areceives a normally logic high HVAUTO input at pin 2 and a normallylogic high HV ON signal input at pin 3. The inputs HVMAN and HVAUTO areconnected to the switch 37 so that they are mutually exclusive. Thesignal input HV ON is provided by the controller 32. In the preferredembodiment of the present invention, the switch 37 also has an OFFposition and may be, by way of example, a C&K Model 7103. When switch 37is in the OFF position, HVMAN and HVAUTO are both at a logic high. Thecondition where HVMAN is a logic low represents selection by a humanoperator to operate the system 10 under direct human supervision in amanual mode by positioning the switch 37 in the MAN position. Thecondition where HVAUTO is a logic low represents selection by a humanoperator to operate the system 10 under supervision of the controller 32by positioning the switch 37 in the AUTO position.

The operation of the HV interlock circuit 182 is described withreference to FIG. 10. Assume the case where: 1) a human operator selectsswitch 37 so as to operate the system 10 in the automatic mode so thatHVMAN is a logic high and HVAUTO is a logic low; and 2) that the LIDoutput of the light detector circuit 300 is a logic high, representingthat no ambient light penetrates into the light tight body 20. Asdescribed with reference to step 206 (FIG. 8), the controller 32 enablesthe HV power supply 180 and photomultiplier 162, as well as the serialport 35. A logic high provided to the PRESET input of the flipflop U22Aallows the positive to negative transition at pin 1 of the flipflop tocause the Q output to go low. In such case, the logic level at pin 12 ofNOR gate U21D is also low. Given that the inputs to pins 5 and 6 arehigh and low, respectively, the output of NOR gate U21B is a logic low.Since the inputs at pins 2 and 3 are both low, the output of NOR gateU21A is a logic high. The input to pins 8 and 9 then are a logic low andhigh, respectfully, resulting in a logic low output which is provided topin 11 of NOR gate U21D and to the clock input of the flipflop U22A. Theinputs on pins 11 and 12 of NOR gate U21D are then both low. Therefore,the output of NOR gate U21D is a logic high which is fed to invertingbuffer U20, thereby generating a complementary HV GATE output which isused to enable the photomultiplier tube 162.

If the output of the light detecting circuit 300 becomes a logic low,indicating ambient light entering the light tight chamber 20, the lowlogic level provided to the PRESET input of the flipflop U22A, causesthe output signal "Q" to be high. Thus, pin 12 of NOR gate U21D is highwhile the input at pin 11 of NOR gate U21D remains a logic low. Thislatter condition causes the outputs of NOR gate U21D, and hence thecomplement of HV GATE to be low. When the complement of HVgate is low,the photomultiplier tube 162 is disabled. Should the LID output providedto the flipflop U22A change from a low state back to a high state, theinputs at pins 11 and 12 of NOR gate U21D will both remain at theirpresent logic state even though pin 11 sees a logic low and pin 12 seesa logic high. Therefore, the output of NOR gate U21D will remain a logiclow so that the photomultiplier tube 162 remains disabled. In order toenable the photomultiplier tube 162, the HVAUTO signal must change froma logic low to a logic high, and back to a logic low again whereupon theoutput of NOR gate U21A changes from logic high to a logic low. Thechange in the logic level at the output of NOR gate U21C (pin 10) maychange from a logic low to a logic high and then back to a logic low bytoggling switch 37 through the OFF position so that the CLK input offlipflop U22A sees the falling edge of the changing logic level at pin10. When the CLK input of the flipflop U22A sees the falling edge of thelogic signal at pin 1 of the flipflop, and if the input to the PRESET ofthe flipflop is a logic high, the output Q becomes a logic low. Now, thelogic states of both pins 11 and 112 are low so that the output of NORgate U21D is a logic high, thereby causing the complementary HV GATEsignal to enable the photomultiplier tube 162. Thus, it may beappreciated that the HV interlock circuit 182 provides the importantsafety function whereby the detection of light by the light detectingcircuit 300 causes the photomultiplier tube 162 to become disabled andremain so until after the switch 37 is toggled manually, as describedabove.

A suitable bioluminescent species which may be used in sample solutionsin conjunction with the present invention is Gonyalaux polyedra becauseit is easy to culture, hardy, and has a light output which is sensitiveto a variety of toxicants. This species may be maintained in enrichedseawater medium (ESM) according to American Society for Testing andMaterials Standard Guide for Conducting Static 96-h Toxicity Tests withMicroalgae (ASTM 1990). Tetrasodium ethylenediaminetetraacetic acid(EDTA), a chelator, may be removed from preparation of the ESM duringassays. Cultures were maintained in 250-mL glass flasks under a lightregime of 12:12 (light:dark) at approximately 4000 LUX from cool whitebulbs. Gonyalaux polyedra was maintained at 18° C. in a controlledtemperature bath. Typically, Gonyalaux polyedra was cultured at 6 to8×10³ cells/mL.

Obviously, many modifications and variations of the present inventionare possible in light of the above teachings. For example, the lightdetecting element in the light detector system 30 may be implemented asa vacuum avalanche diode or as a blue enhanced photodiode array. It istherefore to be understood that within the scope of the appended claims,the invention may be practiced otherwise than as specifically described.

We claim:
 1. A system for measuring toxicity levels in an aqueoussolution of light emitting organisms, comprising:a water proof samplecontainer transparent to visible light; light tight chamber means havinga cavity for holding said sample container and having a light port;first means positioned in said sample container for generating pressurepulses in an aqueous solution; a light detector system optically coupledto detect light emissions generated in sample container which propagatethrough said light port, and mounted to said light tight chamber meansin a light tight manner for generating an electric pulse in response todetecting each of said detected light emissions; and a controller forcounting said electric pulses within a predetermined period of time andfor enabling said first means and said light detector system.
 2. Thesystem of claim 1 further including:a switch operably coupled to saidoptical detector system; and second means for disabling said lightdetector system when ambient light penetrates said light tight chambermeans to prevent said controller from enabling said optical detectingsystem until said switch is manually toggled.
 3. The system of claim 2wherein said second means includes a light emitting diode and aphototransistor.
 4. The system of claim 1 which includes:an electricmotor mounted to said light tight chamber means; and said first meansincludes an output shaft powered by said electric motor and whichextends into said sample container, and a propeller mounted to saidoutput shaft so as to be positioned within said sample container.
 5. Thesystem of claim 4 where said controller selectively enables saidelectric motor.
 6. The system of claim 1 which further includes anoptical filter interposed between said sample container and said lightdetector system.
 7. The system of claim 1 wherein said optical detectorsystem is mounted in a magnetic shielded enclosure.
 8. The system ofclaim 1 wherein said sample container holds an aqueous solution ofbioluminescent organisms.
 9. The system of claim 8 wherein saidbioluminescent organisms include Gonvalaux polyedra.
 10. The system ofclaim 1 further including a lift mechanism for removing said samplecontainer from said light tight chamber means.
 11. A system formeasuring toxicity levels in an aqueous solution of light emittingorganisms, comprising:a sample container transparent to visible lightfor holding an aqueous solution of bioluminescent organisms; light tightchamber means having a cavity for holding said sample container andhaving a light port; first means for generating pressure pulses in saidaqueous solution to stimulate said organisms to generate lightemissions; a light detector system optically coupled to detect saidlight emissions through said light port and mounted to said light tightchamber means in a light tight manner for generating an electric pulsein response to detecting each of said detected light emissions; and acontroller for counting said electric pulses within a predeterminedperiod of time and for enabling said first means and said light detectorsystem.
 12. The system of claim 11 further including:a switch operablycoupled to said optical detector system; and second means for disablingsaid light detector system when ambient light penetrates said lighttight chamber means to prevent said controller from enabling saidoptical detecting system until said switch is manually toggled.
 13. Thesystem of claim 12 wherein said second means includes a light emittingdiode and a phototransistor.
 14. The system of claim 11 whichincludes:an electric motor mounted to said light tight chamber means;and said first means includes an output shaft powered by said electricmotor and which extends into said sample container, and a propellermounted to said output shaft so as to be positioned within said samplecontainer.
 15. The system of claim 14 where said controller selectivelyenables said electric motor.
 16. The system of claim 11 which furtherincludes an optical filter interposed between said sample container andsaid light detector system.
 17. The system of claim 11 wherein saidoptical detector system is mounted in a magnetic shielded enclosure. 18.The system of claim 11 wherein said sample container holds an aqueoussolution of bioluminescent organisms.
 19. The system of claim 18 whereinsaid bioluminescent organisms include Gonvalaux polyedra.
 20. The systemof claim 11 further including a lift mechanism for removing said samplecontainer from said light tight chamber means.
 21. The system of claim11 wherein said controller waits a predetermined period of time afterenabling said optical detecting system before counting said pulses.